In accordance with the present invention, it has been discovered that various members of the steroid/thyroid superfamily of receptors can interact to form multimeric species comprising a complex of more than one receptor. Accordingly, the interaction of a first receptor species with a second receptor species modulates the ability of the first receptor species to trans-activate transcription of genes maintained under hormone expression control in the presence of the cognate ligand for said first receptor.
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22. A dimeric receptor comprising at least the dimerization domain of one isoform of RXR and a different member of the steroid/thyroid hormone superfamily of receptors.
19. A dimeric receptor comprising one isoform of RXR and at least the dimerization domain of a different member of the steroid/thyroid hormone superfamily of receptors.
1. A dimeric receptor comprising one member selected from isoforms of RXR and one different member of the steroid/thyroid superfamily of receptors, wherein said members are associated in the form of a dimer.
2. A receptor according to
3. A dimeric receptor according to
4. A dimeric receptor according to
5. A receptor according to
6. A receptor according to
9. A receptor according to
10. A receptor according to
20. A dimeric receptor according to
21. A dimeric receptor according to
23. A dimeric receptor according to
24. A dimeric receptor according to
25. A dimeric receptor according to
26. A dimeric receptor according to
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This application is a continuation of application U.S. Ser. No. 08/169,969, filed Dec. 20, 1993, now abondoned, which is a continuation of application U.S. Ser. No. 07/803,163, filed Dec. 6, 1991, now abondoned, the entire contents of which are hereby incorporated by reference herein.
The present invention relates to interactions between members of the steroid/thyroid superfamily of receptor proteins, novel combinations of various members of the steroid/thyroid superfamily of receptor proteins, and methods of using such combinations.
Transcriptional regulation of development and homeostasis in complex eukaryotes, including humans and other mammals, birds, fish, insects, and the like, is controlled by a wide variety of regulatory substances, including steroid and thyroid hormones. These hormones exert potent effects on development and differentiation of phylogenetically diverse organisms. The effects of hormones are mediated by interaction with specific, high affinity binding proteins referred to as receptors.
A number of receptor proteins are known, each specific for steroid hormones [e.g., estrogens (estrogen receptor), progesterones (progesterone receptor), glucocorticoid (glucocorticoid receptor), androgens (androgen receptor), aldosterones (mineralocorticoid receptor), vitamin D (vitamin D receptor)], retinoids (e.g., retinoic acid receptor) or thyroid hormones (e.g., thyroid hormone receptor. Receptor proteins have been found to be distributed throughout the cell population of complex eukaryotes in a tissue specific fashion.
Molecular cloning studies have made it possible to demonstrate that receptors for steroid, retinoid and thyroid hormones are all structurally related and comprise a superfamily of regulatory proteins. These regulatory proteins are capable of modulating specific gene expression in response to hormone stimulation by binding directly to cis-acting elements.
An important advance in the characterization of this superfamily of regulatory proteins has been the identification of a growing list of gene products which possess the structural features of hormone receptors.
It is known that steroid or thyroid hormones, protected forms thereof, or metabolites thereof, enter cells and bind to the corresponding specific receptor protein, initiating an allosteric alteration of the protein. As a result of this alteration, the complex of receptor and hormone (or metabolite thereof) is capable of binding with high affinity to certain specific sites on chromatin.
It is also known that many of the primary effects of steroid and thyroid hormones involve increased transcription of a subset of genes in specific cell types.
A number of transcriptional control units which are responsive to members of the steroid/thyroid superfamily of receptors have been identified. These include the mouse mammary tumor virus 5′-long terminal repeat (MTV LTR), responsive to glucocorticoid, aldosterone and androgen hormones; the transcriptional control units for mammalian growth hormone genes, responsive to glucocorticoids, estrogens and thyroid hormones; the transcriptional control units for mammalian prolactin genes and progesterone receptor genes, responsive to estrogens; the transcriptional control units for avian ovalbumin genes, responsive to progesterones; mammalian metallothionein gene transcriptional control units, responsive to glucocorticoids; and mammalian hepatic α2u-globulin gene transcriptional control units, responsive to androgens, estrogens, thyroid hormones, and glucocorticoids.
A major obstacle to further understanding and more widespread use of the various members of the steroid/thyroid superfamily of hormone receptors has been a lack of awareness of the possible interactions of various members of the steroid/thyroid superfamily of hormone receptors, and an understanding of the implications of such interactions on the ability of members of the steroid/thyroid superfamily of hormone receptors to exert transcriptional regulation of various physiological processes.
In accordance with the present invention, we have discovered that two or more members of the steroid/thyroid superfamily of receptors can combine to form multimeric species comprising a complex of more than one receptor. Accordingly, the combination of a first receptor species with a second receptor species is capable of modulating the ability of the first receptor species to trans-activate transcription of genes maintained under expression control in the presence of cognate ligand for said first receptor.
In accordance with the present invention, there are provided multimeric receptor species which belong to the steroid/thyroid superfamily of receptors, comprising at least the dimerization domain of at least two different members of the steroid/thyroid superfamily of receptors.
As employed herein, the term “dimerization domain” of a member of the steroid/thyroid superfamily of receptors refers to that portion of the receptor which is believed to be involved in the formation of multimeric receptor species. This domain typically comprises the carboxy-terminal portion of the receptor, i.e., that portion of a receptor which is 3′ with respect to the DNA-binding domain of the receptor.
In accordance with the present invention, there are also provided combination(s) of receptors comprising at least two different members of the steroid/thyroid superfamily of receptors, wherein said receptors are associated in the form of a multimer;
Combinations contemplated by the present invention can broadly be referred to as “multimeric species”, which is intended to embrace all of the various oligomeric forms in which members of the steroid/thyroid superfamily of receptors (including fragments thereof comprising the dimerization domains thereof) are capable of associating. Thus, reference to “combinations” of steroid receptors or “multimeric” forms of steroid receptors includes homodimeric combinations of a single receptor (including fragments thereof comprising the dimerization domains thereof), heterodimeric combinations of two different receptors (including fragments thereof comprising the dimerization domains thereof), homotrimeric combinations of a single receptor (including fragments thereof comprising the dimerization domains thereof), heterotrimeric combinations of two or three different receptors (including fragments thereof comprising the dimerization domains thereof), homotetrameric combinations of a single receptor (including fragments thereof comprising the dimerization domains thereof), heterotetrameric combinations of two or more different receptors (including fragments thereof comprising the dimerization domains thereof), and the like.
As employed herein, the phrase “members of the steroid/thyroid superfamily of receptors” refers to all of the various isoforms of hormone binding proteins that operate as ligand-dependent transcription factors, including members of the steroid/thyroid superfamily of receptors for which specific ligands have not yet been identified (referred to hereinafter as “orphan receptors”). Each such protein has the intrinsic ability to bind to a specific DNA sequence in a target gene. Following binding, the transcriptional activity of the gene is modulated by the presence or absence of the cognate hormone (ligand).
The DNA-binding domains of all members of this superfamily of receptors are related, consisting of 66-68 amino acid residues, and possessing about 20 invariant amino acid residues, including nine cysteines. A member of the superfamily can be identified as a protein which contains these diagnostic amino acid residues, which are part of the DNA-binding domain of such known steroid receptors as the human glucocorticoid receptor (amino acids 421-486), the estrogen receptor (amino acids 185-250), the mineralocorticoid receptor (amino acids 603-668), the human retinoic acid receptor (amino acids 88-153), and the like. The highly conserved amino acids of the DNA-binding domain of members of the superfamily are as follows:
Exemplary members of the steroid/thyroid superfamily of receptors (including the various isoforms thereof) include steroid receptors such as glucocorticoid receptor, mineralocorticoid receptor, progesterone receptor, androgen receptor, vitamin D3 receptor, and the like; plus retinoid receptors, such as the various isoforms of RAR (e.g., RARα, RARβ, or RARγ), the various isoforms of RXR (e.g., RXRα, RXRβ, or RXRγ), and the like; thyroid receptors, such as TRα, TRβ, and the like; as well as other gene products which, by their structure and properties, are considered to be members of the superfamily, as defined hereinabove, including the various isoforms thereof. Examples of orphan receptors include hepatocyte nuclear factor 4, hereinafter referred to as HNF4 ([see, for example, Sladek et al., in Genes & Development 4: 2353-2365 (1990)], the COUP family of receptors [see, for example, Miyajima et al., in Nucleic Acids Research 16: 11057-11074 (1988), and Wang et al., in Nature 340: 163-166 (1989)], COUP-like receptors and COUP homologs, such as those described by Mlodzik et al., in Cell 60: 211-224 (1990) and Ladias et al., in Science 251: 561-565 (1991), various isoforms of peroxisome proliferator-activated receptors hereinafter referred to as (PPARs; see, for example, Issemann, and Green, in Nature 347: 645-650 (1990)), the ultraspiracle receptor hereinafter referred to as usp [see, for example, Oro et al., in Nature 347: 298-301 (1990)], and the like. Presently preferred members of the superfamily for use in the practice of the present invention are those members which recognize “direct repeat” hormone response elements, as described in detail hereinbelow.
The formation of multimeric species can modulate the ability of the first receptor to trans-activate transcription of genes maintained under expression control in the presence of ligand for said first receptor. The actual effect on activation of transcription (i.e., enhancement or repression of transcription activity) will vary depending on the receptor species which are part of the multimeric species, as well as on the response element with which the multimeric species interacts. Thus, for example, formation of a heterodimer of RXR and RAR inhibits the ability of RXR to trans-activate RXR-mediated processes, while the same heterodimer provides enhanced trans-activation activity with respect to the ability of RAR to trans-activate RAR-mediated processes.
In accordance with another embodiment of the present invention, there is provided a method to modulate, in an expression system, the transcription activation of a gene by a first member of the steroid/thyroid superfamily of receptors, wherein the expression of said gene is maintained under the control of a hormone response element, said method comprising:
As employed herein, the term “modulate” refers to the ability of a given multimeric species to either enhance or repress a receptor's ability to induce transcription of a target gene, relative to such ability of said receptor in its uncomplexed state. The actual effect of multimerization on a receptor's transcription activity will vary depending on the specific receptor species which are part of the multimeric species, and on the response element with which the multimeric species interacts. Thus, for example, formation of a heterodimer of RXR and TR inhibits the ability of RXR to trans-activate RXR-mediated processes, while the same heterodimer provides enhanced trans-activation activity with respect to the ability of TR to trans-activate TR-mediated processes.
In accordance with one embodiment of the present invention, the first member of the steroid/thyroid superfamily of receptors is an isoform of RXR and the second member is selected from COUP-TF, EAR-2, PPAR, VDR, TR, RAR, or isoforms thereof. Those of skill in the art can readily identify the compound(s) and/or condition(s) which induce expression of one or more of the second members set forth above.
In accordance with this embodiment, the first member is encoded by a gene expressed in the liver, spleen, kidney, and/or small intestine. The encoded product(s) are involved in lipid metabolism and/or cholesterol homeostasis.
In accordance with another embodiment of the present invention, the first member of the steroid/thyroid superfamily of receptors is an isoform of RAR and said second member is an isoform of RXR. Those of skill in the art can readily identify the compound(s) and/or condition(s) which are capable of inducing expression of one or more isoforms of the second member (RXR) as set forth above.
In accordance with still another embodiment of the present invention, the first member of the steroid/thyroid superfamily of receptors is an isoform of TR and the second member is an isoform of RXR. Those of skill in the art can readily identify the compound(s) and/or condition(s) which are capable of inducing expression of one or more isoform of the second member (RXR) as set forth above.
In accordance with yet another embodiment of the present invention, the first member of the steroid/thyroid superfamily of receptors is VDR and the second member is an isoform of RXR. Those of skill in the art can readily identify the compound(s) and/or condition(s) which are capable of inducing expression of one or more isoform of the second member (RXR) as set forth above.
Hormone response elements contemplated for use in the practice of the present invention include naturally occurring response elements, or synthetic response elements which are composed of two or more “half sites”, wherein each half site comprises the sequence
-RGBNNM-,
wherein
When one of the half sites varies by 2 nucleotides from the preferred sequence of -AGGTCA-, it is preferred that the other half site of the response element be the same as, or vary from the preferred sequence by no more than 1 nucleotide. It is presently preferred that the 3′-half site (or downstream half site) of a pair of half sites vary from the preferred sequence by at most 1 nucleotide.
Since the half sites are combined in direct repeat fashion (rather than as palindromic constructs), the resulting synthetic response elements are referred to as “DR-x”, wherein “DR” refers to the direct repeat nature of the association between the half sites, and “x” indicates the number of spacer nucleotides between each half site.
Exemplary response elements useful in the practice of the present invention are derived from various combinations of half sites having sequences selected from, for example, -AGGTCA-, -GGTTCA-, -GGGTTA-, -GGGTGA-, -AGGTGA-, -GGGTCA-, and the like.
The nucleotides employed in a non-zero spacer are independently selected from C, T, G, or A.
Exemplary three nucleotide spacers include -AGG-, -ATG-, -ACG-, -CGA-, and the like. Exemplary four nucleotide spacers include -CAGG-, -GGGG-, -TTTC-, and the like. Exemplary five nucleotide spacers include -CCAGG-, -ACAGG-, -CCGAA-, -CTGAC-, -TTGAC-, and the like.
Exemplary response elements contemplated by the present invention include the following DR-3 elements:
Presently preferred response elements contemplated for use in the practice of the present invention include:
The invention will now be described in greater detail by reference to the following non-limiting examples.
Plasmids
Receptor expression plasmids used in the cotransfection assays are described by Mangelsdorf et al. [see Cell Vol. 66:555-561 (1991)]; and Umesono et al. [see Cell Vol. 65:1-20 (1991)].
RS-COUP-TF expression plasmid was constructed by inserting an Asp718-BamHI fragment containing the EAR-3 (i e., COUP) coding region [Miyajima et al., Nucl. Acids Res. Vol. 16:11057-11074 (1988)] into Asp718-BamHI-cut pRS expression vector.
To construct the RS-EAR-2 expression plasmid, an Eco47III-BglII fragment containing the EAR-2 coding region (Miyajima et al., supra) was blunted with Klenow and inserted into Asp718-BamHI-cut pRS, which had also been end-filled with Klenow.
All of the recombinant reporter constructs used contain either one or two copies of the indicated oligonucleotides inserted at the unique HindIII site upstream of the basal reporter construct ΔSV-CAT (Umesono et al., supra). Identity and orientation of the inserted oligonucleotides was confirmed by sequencing.
Cotransfection Assays
CV-1, HeLa, and F9 teratocarcinoma cell culture, transfections, and CAT assays were performed as previously described (Mangelsdorf et al., supra: Umesono et al., supra). In cotransfection experiments including expression plasmids RS-COUP-TF and RS-EAR-2 (see FIG. 2), cell extracts were normalized to total amount of protein for use in CAT assays, as these expression constructs were shown to severely repress expression of β-galactosidase expression vectors.
Bacterial Expression of RXR and COUP-TF
hRXRα was expressed in bacteria as a fusion protein with glutathione-S-transferase using the pGEX-2T expression vector [Smith and Johnson, Gene Vol. 67:31-40 (1988)]. Purification of the fusion protein and cleavage of the glutathione-S-transferase protein from RXR with thrombin were performed as described by Mangelsdorf et al., supra.
For expression of COUP-TF in bacteria, a 1.8 kb NcoI-BamHI fragment containing the entire coding region of EAR-3 (Miyajima et al., supra) was inserted into the PET-8C expression vector [Studier et al., Methods in Enzymology 185: 60-89 (1990). BL21(DE3)plysS cells [Studier et al., supra] containing the PET-8C-COUP-TF expression construct were induced for 3 hours with 0.6 mM isopropylthiogalactoside (IPTG) and the cells subsequently lysed in lysis buffer [50 mM Tris (pH 8.0), 250 mM KCl,1 mM DTT, 1 mM PMSF, 1% Triton X-100] by freeze-thawing.
Lysates were clarified by centrifugation for 1 hour at 45,000 rpm in a Ti60 rotor (Beckman). Crude bacterial lysates containing COUP-TF were diluted in lysis buffer lacking KCl to a final concentration of 100 mM KCl and loaded on a heparin-agarose column. The column was washed with Buffer A [20 mM Tris (pH 8.0), 20% glycerol, 1 mM DTT, 1 mM PMSF], and COUP-TF subsequently eluted with Buffer A containing 800 mM KCl.
The eluted protein was dialyzed to 100 mM KCl, loaded on a MonoQ column (Pharmacia), and protein eluted with a linear salt gradient (100 mM-800 mM) in Buffer A. Fractions containing COUP-TF binding activity (eluting at 300-350 mM KCl) were pooled and aliquoted for use in gel mobility shift assays. Western blot analysis done using COUP-TF-specific antiserum confirmed that the partially-purified COUP-TF migrated upon SDS-PAGE as an ˜45 kD protein.
DNA-Binding Assays
Gel mobility shift assays (20 μl) contained 10 mM Tris (pH 8.0), 40 mM KCl, 0.1% NP-40, 6% glycerol, 1 μg of poly(dI-dC), and the specific receptor species indicated in the figure legends. After 10 minutes incubation on ice, 1 ng of 32P-labeled oligonucleotide was added and the incubations were continued for an additional 10 minutes. DNA-protein complexes were resolved on 4% polyacrylamide gels in 0.5×TBE (1×TBE=90 mM Tris, 90 mM boric acid, 2 mM EDTA). Gels were dried and subjected to autoradiography at −70° C. The following oligonucleotides and their complements were 32P-labeled and used as probes:
ID No. 26);
A peptide corresponding to amino acids 214-229 of hRXRα was synthesized according to the technique of Rivier et al. [Science Vol. 224:889-891 (1984)]. A glycine and tyrosine were added to the carboxy terminus for coupling to human α-globulins using bisdiazotized benzadine as described by Vaughan et al., in Methods in Enzymology Vol. 168:588-617 (1989). For initial injection, Freund's complete adjuvant was mixed with an equal volume of physiological saline containing 1 mg conjugate/ml. For boosters, Freund's incomplete adjuvant was mixed with an equal volume of physiological saline containing 0.5 mg conjugate/ml. For each immunization, a rabbit received a total of 1 ml emulsion in multiple intradermal sites. Animals were injected every three weeks and bled through an ear vein seven days after each boost. Serum was collected and evaluated for receptor antibodies on the basis of Western blot analysis of hRXRα transfected COS cell extracts. The antisera used herein was collected after the sixth boost.
Bacterial-expressed COUP-TF and RXR-glutathione-S-transferase fusion protein (RXR-GST) were mixed and the resulting complexes analyzed by gel mobility shift assays using 32P-labeled DR-1 oligonucleotide (i e., SEQ ID No. 21) as probe. The larger RXR fusion protein was used in order to maximize the migratory differences observed between the COUP-TF and RXR complexes. RXR-GST behaved identically to the nonfusion protein in terms of binding specificity with all the response elements tested, including exhibiting a marked preference for DR-1 relative to the other DRs.
Gel mobility shift assays were performed using 32P-labeled DR-1 oligonucleotide (SEQ ID No. 21) in the presence of partially-purified COUP-TF (500 ng) and increasing amounts of partially-purified RXR (1×-50 ng) as indicated in FIG. 1. Either 0.3 μl or 1 μl of RXR-specific antiserum was included in the assays (shown in lanes 11 and 12, respectively). The positions of the RXR-specific and COUP-TF-specific complexes are indicated in
As shown in
Addition of RXR-specific antiserum to an assay containing both COUP-TF and RXR-GST resulted in the “supershifting” of the COUP-TF-RXR complex (lane 11). The RXR-specific antiserum did not cross-react with bacterially-expressed COUP-TF. Increasing the amount of antiserum added to the gel mobility shift assay ultimately resulted in the disruption of the COUP-TF-RXR interaction and reappearance of the COUP-TF-specific complex (lane 12). The release of COUP-TF from this complex is a likely consequence of higher amounts of the antibody stabilizing RXR homodimers.
Similar supershift data, indicating the formation of a COUP-TF-RXR heterodimeric complex, were also obtained using radiolabeled ovalbumin COUP-TF RE as probe. These results, taken together, provide compelling evidence that COUP-TF and RXR can form a highly stable heterodimeric complex in vitro.
The observation that RXR can stimulate transcription through a COUP-TF recognition element suggests that COUP-TF might reciprocally activate through a CRBPII site. The in vitro binding data presented above strongly supports this proposal. However, in cotransfection analyses, it is not possible to obtain a significant COUP-TF-mediated activation of expression from reporter plasmids COUP RE2-ΔSV-CAT or CRBPII-ΔSV-CAT when tested in either F9, CV-1, or HeLa cells (
As an alternative approach, it was investigated whether COUP-TF could alter RXR-mediated induction from the CRBPII-RXRE. Accordingly, the CRBPII-CAT reporter, containing the intact promoter region of the CRBPII gene, was cotransfected into F9 cells with either RXR expression plasmid alone, or in combination with expression plasmids for either COUP-TF or EAR-2. F9 cells were cotransfected in duplicate with 3 μg the reporter pCRBPII-CAT and 1 μg of RS-hRXRα plus 0.5 μg of either the control RS-LUC (lanes 1 and 2), RS-hRARα (lanes3 and 4), RS-COUP-TF (lanes 5 and 6), or RS-EAR-2 (lanes 7 and 8). Transfection of each 10 cm plate also included 5 μg of RAS-β-galactosidase and 5.5 μg of pUC19 as carrier. Cotransfections performed with the reporter pCRBPII-CAT and either 0.5 μg RS-COUP-TF (lanes 9 and 10) or 0.5 μg of RS-EAR-2 (lanes 11 and 12) in the absence of RS-hRXRα are also shown in FIG. 2. Cells were treated with either ethanol (−) or 10 μM RA (+) for 30 hours and the cell extracts subsequently assayed for CAT activity. One set of the duplicate CAT assays is shown in the Figure.
As expected, addition of retinoic acid (RA) to cells cotransfected with CRBPII-CAT reporter and RXR expression plasmid resulted in a dramatic (approximately 90-fold) induction of CAT activity (
To demonstrate that this repression was mediated by the CRBPII element, a parallel experiment utilizing the CRPBII-ΔSV-CAT reporter was performed in CV-1 cells. CV-1 cells were cotransfected in duplicate with the reporter CRBPII-ΔSV-CAT and RS-hRXRα (1μg) in the presence of 0.5 μg RS-LUC [as a control; designated in the figure as (C)], or 0.2 and 0.5 μg of RS-COUP-TF or RS-EAR-2. Cells were treated with either ethanol (−) or 10 μM RA (+) and the cell extracts subsequently assayed for CAT activity. CAT activity is shown in
Similar results were obtained, with both COUP-TF and EAR-2 functioning as potent inhibitors of RXR-mediated activation (FIG. 2B). As shown in
These results indicate that COUP-TF/EAR-2-mediated suppression of reporter activity is specific for RXR and its response element.
Immunoprecipitation experiments were performed using bacterially-expressed RXR and 35S-methionine-labeled RAR synthesized in vitro. RAR, LBD, or GR RNA was prepared and subsequently translated in rabbit reticulocyte lysates as directed by the supplier (Promega). RXR was expressed in bacteria as a fusion with glutathione-S-transferase using the pGEX-2T expression vector (Pharmacia) as described by Mangelsdorf et al., supra. Immunoprecipitation reactions (20 μl) included 5 μl of [35S]methionine-labeled receptor protein and 150 ng of either purified GST-RXR or GST alone in 20 mM Tris, pH 8.0. Proteins were incubated 20 minutes on ice prior to the addition of 5 μl of polyclonal RXR antiserum. Antigen-antibody complexes were collected by the addition of Protein A-Sepharose (Pharmacia) and the immunocomplexes washed three times with 400 μl RIPA buffer [10 mM Tris (pH 8.0), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate]. Immunoprecipitated complexes were resolved by SDS polyacrylamide gel electrophoresis on 10% gels which were then fixed in 30% methanol, 10% acetic acid, dried, and subjected to autoradiography. Gel retardation assays (20 μl) contained 10 mM Tris (pH 8.0), 40 mM KCl, 0.1% NP-40, 6% glycerol, 0.2 mM EDTA, 0.1 mM DTT, 0.2 μg of poly(dI-dC) and 2.5 μl of in vitro synthesized RAR and RXR proteins. When either RAR or RXR was omitted, the reaction was supplemented with the same volume of unprogrammed reticulocyte lysate. After a 10 minute incubation on ice, 1 ng of 32P-labeled oligonucleotide was added and the incubation continued for an additional 10 minutes. DNA-protein complexes were resolved on a 4% polyacrylamide gel in 0.5×TBE (1×TBE=90 mM Tris, 90 mM boric acid, 2 mM EDTA). Gels were dried and subjected to autoradiography at −70°. Gel mobility shift assays performed using Cos cell-expressed receptors were performed as described by Umesono et al., supra using whole cell extracts prepared from Cos cells transfected with either RS-hRARa, RS-hRXRa, or both expression plasmids.
As shown in
Similar experiments in which RAR was replaced with radiolabeled GR failed to reveal RXR-GR interactions, demonstrating the specificity of the RAR-RXR interaction under these conditions (see
The stability of the RAR-RXR heterodimer in solution suggested that the two proteins might also interact and display novel properties when associated with DNA. To test this possibility, gel mobility shift experiments were first performed using in vitro synthesized RAR and RXR and a radiolabeled oligonucleotide encoding the CRBPII-RXRE (i e., SEQ ID No. 28). As shown in
The results of the transfection analyses presented above indicate that, under the conditions employed, the RAR-RXR heterodimer is transcriptionally inactive on the CRBPII-RXRE.
The specificity of the RAR-RXR interaction with DNA was next examined using unlabeled oligonucleotides as competitor. Oligonucleotides containing the CRBPII-RXRE (SEQ ID No. 28) competed efficiently for RAR-RXR heterodimer binding at a 10-fold molar excess (
To further investigate this interaction of the RAR-RXR heterodimer with the βRARE (i e., SEQ ID No. 26), oligonucleotides containing the βRARE were labeled and used as probe in a gel mobility shift assay. As in the case of the CRBPII-RXRE, both in vitro synthesized RAR and RXR were required for high affinity DNA-protein interactions with the βRARE (
Similar results indicating a requirement for the presence of both RAR and RXR for formation of a high affinity DNA-protein complex on the βRARE were obtained using whole-cell extracts prepared from Cos cells which had been transfected with either RAR alone, RXR alone, or both RAR and RXR (FIG. 3E). Taken together, these results demonstrate that RXR dramatically enhances the binding affinity of RAR to a strong retinoic acid response element, and that the RAR-RXR complex is likely to be present in vivo.
Similarly, in immunoprecipitation experiments, in vitro synthesized thyroid receptor-beta (TRβ) and vitamin D receptor (VDR) were found to co-precipitate with bacterially-expressed RXR (
Taken together, these data strongly suggest a central role for members of the steroid/thyroid superfamily of receptors, such as RXR, in modulating the hormonal responses conferred via the RAR, TR, and VDR.
While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.
Evans, Ronald M., Kliewer, Steven A., Umesono, Kazuhiko
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